PROJECT SUMMARY T cells require high and sustained levels of Ca2+ signaling for activation. Ca2+ signaling in T cells is primarily mediated by CRAC (Ca2+ release-activated Ca2+) channels consisting of the pore subunits, Orai proteins, and an endoplasmic reticulum (ER)-resident signaling molecule, STIM1. STIM1 senses ER Ca2+ store depletion triggered by T cell receptor (TCR) stimulation and gates Orai channels. Orai family members, Orai1, 2, and 3 share similar structure and are able to mediate CRAC currents, but with distinct biophysical properties. Mutations in ORAI1 cause severe combined immune deficiency (SCID) in human patients, partly due to its predominant role in effector T cells. Therefore, Orai1 has received high attention due to its potential as a therapeutic target to suppress autoimmunity (e.g. psoriasis). However, a fundamental problem in these approaches is that the current small molecule blockers for Orai1 also inhibit Orai2 and Orai3, which can result in pleiotropic side effects. There is a clear lack of understanding of the specific functions of Orai1, Orai2 and Orai3, which is necessary to develop small molecule blockers specifically targeting each of them. We find that nave T cells predominantly express Orai2 and Orai1 deficiency in nave T cells mildly reduces SOCE. On the contrary, Orai1 knockout effector T cells exhibit pronounced reduction in SOCE, demonstrating its predominant role in effector T cells. These cells have a significant residual Ca2+ entry, which is likely mediated by Orai2 based on pharmacological studies. Therefore, our main hypothesis is that Orai2 plays a predominant role in nave T cells while both Orai1 and Orai2 are important for effector functions of T cells. We will validate this hypothesis using transgenic animal models deleted for ORAI2 alone or both ORAI1 and ORAI2 genes. This study will dissect the contribution of T cell- enriched Orai channel(s) in different phases of development, homeostasis, differentiation, and pathogenicity of T cells. This study is significant because there have been numerous efforts to identify Orai1 inhibitors for therapeutic usage, but the effect of these inhibitors on Orai2 activity has not even been considered. This study to uncover the physiological outcomes of selective block of Orai2 or both Orai1 and Orai2 is important for future therapeutic applications to suppress T cell-mediated inflammatory diseases (e.g., psoriasis) and transplant rejection, or to enhance efficacy of T cell-mediated cancer therapy. !